The present invention relates to reagents useful in the quantitative determination of proteolytic enzymes. More particularly the present invention relates to peptide derivatives which are substrates for enzymes of the class E.C.3.4.4. These enzymes cleave amide linkages in peptide chains on the carbonyl side of arginine and lysine residues.
Classical substrates for trypsin, thrombin, and related enzymes have involved amides such as N.sup..alpha. -benzoyl-DL-arginyl-p-nitroanilide, L-lysyl-p-nitroanilide, N.sup..alpha. -benzoyl-DL-arginyl-2-naphthylamide and other di, tri, and higher order arginine and lysine peptides with chromogenic amide leaving groups [B. F. Erlanger, et al., Arch. Biochem. Biophys., 95, (1961) 271; A. Riedel and E. Wunsch, Z. Physiol. Chem., 316, (1961) 1959; R. E. Plapinger, et al., J. Org. Chem., 30 (1965) 1781; L. Svendsen, et al.].
The advantage of extending the amino terminal end of either arginyl or lysyl-p-nitroanilide substrates results in improved substrate behavior [Thrombosis Res.,1, (1972) 267-78; U.S. Pat. No. 3,884,896], and in particularly, a number of prior art disclosures describes various p-nitroaniline derivatives of tripeptides or higher peptides [U.S. Pat. Nos. 4,070,245; 4,061,625; and 4,016,042].
The present invention reagents are p-nitroaniline derivatives of dipeptides containing a thiomethylene group in place of a peptide linkage. Thiomethylene substituted for the peptide linkage between glycine and leucine in a compound of the formula (S)-2-(S-cysteaminyl)-4-methylpentoic acid is described by K. F. Fok and J. A. Yankeelov, Jr., [Biochem. and Biophys. Res. Comm., 74, 273 (1977)] with the resulting thiomethylene substituted peptide active as an effective inhibitor of aminopeptidase M. The invention reagents, however, are structurally different and act as effective substrates for proteolytic enzymes rather than as enzyme inhibitors.